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WO2016013041A1 - Procédé de détection et dispositif de détection de cellule tumorale circulante - Google Patents

Procédé de détection et dispositif de détection de cellule tumorale circulante Download PDF

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WO2016013041A1
WO2016013041A1 PCT/JP2014/003855 JP2014003855W WO2016013041A1 WO 2016013041 A1 WO2016013041 A1 WO 2016013041A1 JP 2014003855 W JP2014003855 W JP 2014003855W WO 2016013041 A1 WO2016013041 A1 WO 2016013041A1
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cells
immunostaining
antibody
cancer
culture
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Japanese (ja)
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恒雄 倉持
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Institute Of Cancer Immunology Co Ltd
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Institute Of Cancer Immunology Co Ltd
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Priority to JP2016535556A priority Critical patent/JP6052756B2/ja
Priority to KR1020167032240A priority patent/KR101922322B1/ko
Priority to CN201480078553.7A priority patent/CN106796237B/zh
Priority to PCT/JP2014/003855 priority patent/WO2016013041A1/fr
Publication of WO2016013041A1 publication Critical patent/WO2016013041A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/4833—Physical analysis of biological material of solid biological material, e.g. tissue samples, cell cultures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals

Definitions

  • the present invention relates to a peripheral circulating cancer cell detection method and a detection apparatus capable of easily detecting peripheral circulating cancer cells (CTC; Circulating Tumor Cell).
  • CTC peripheral circulating cancer cells
  • peripheral circulating cancer cells are observed in the peripheral blood of patients with epithelial-derived cancer at a single ultra-low concentration in 106-107 peripheral blood mononuclear cells. .
  • CTC is considered to cause advanced cancer cells to circulate on the flow of blood and lymph and cause metastasis to distant organs.
  • CTCs in the blood have been recognized as useful in determining the therapeutic effect and predicting prognosis in cases of metastatic cancer such as breast cancer and colorectal cancer.
  • breast cancer and colorectal cancer developed by Veridex, USA
  • a CellSearch registered trademark
  • CTCs are concentrated and sorted using anti-EpCAM antibody-immobilized magnetic beads, and then CTCs in a sample are counted by identifying the CTCs by immunostaining (for example, (Refer nonpatent literature 1).
  • urokinase activity A method for detecting circulating cancer cells that detects and collects CTCs simply and accurately is known (see Patent Document 1).
  • CTCs are concentrated using anti-EpCAM antibody-immobilized magnetic beads against EpCAM (epithelial cell surface molecule) expressed on the surface of cancer cells.
  • EpCAM epithelial cell surface molecule
  • epithelial cells are specifically separated from many cells in blood by magnetic particles in which antibodies against EpCAM are bound to nano iron particles.
  • the separated epithelial cells are reacted with a fluorescently labeled cytokeratin monoclonal antibody, and the cell nucleus is stained with a fluorescent DNA staining substance.
  • a fluorescently labeled CD45 antibody is reacted.
  • the CTC reaction solution is transferred into a cartridge having a magnet fixed thereto.
  • the CTC moves to the upper surface of the cartridge by the magnetic force generated by the magnet. Fluorescent image data indicating the fluorescent color development state on the upper surface of the cartridge is analyzed.
  • an object of the present invention is to provide a detection method and a detection apparatus that can easily and accurately detect peripheral circulating cancer cells.
  • peripheral cells of the peripheral circulation cancer simply and accurately using the adherent cells after cell culture. It was found that it could be detected.
  • the peripheral circulating cancer cell detection method of the present invention is a method for detecting circulating cancer cells in a biological sample, and includes the following steps (a) to (d).
  • (A) Separation step for separating lymphocytes from peripheral blood (b) Culturing step for culturing the separated peripheral blood lymphocyte layer in a culture solution (c) After culturing, immunostaining the cells attached to the bottom of the culture vessel Immunostaining step (d) Detection step of detecting circulating cancer cells in a biological sample based on an observation image of stained cells obtained by the immunostaining step
  • the immunostaining step (c) is more preferably performed using an anti-EMA (Epithelial® Membrane® Antigen) antibody.
  • anti-EMA Epidermal® Membrane® Antigen
  • the results obtained by immunostaining using anti-EMA antibody are equivalent to lowering the cut-off value of the test because the analysis data has low specificity and high sensitivity. This is because it can be used as a screening test.
  • the culture vessel used in the culture step (b) and the immunostaining vessel used in the immunostaining step (c) are the same vessel, and after culturing, only the cells attached to the bottom by draining the supernatant from the culture vessel It is preferable to inject the antibody to be immunostained into the culture container. This is because it can be detected more easily.
  • the culture solution may contain at least 1000 U / ml interleukin-2 (IL-2).
  • the culture medium for culturing the peripheral blood lymphocyte layer preferably empirically contains at least 1000 U / ml interleukin-2.
  • the culture step (b) is preferably performed for 48 to 72 hours. This is because it is difficult to detect circulatory cancer cells empirically in cultures shorter than 48 hours, and in cancer cultures longer than 72 hours, the circulating cancer cells disappear spontaneously (apoptosis).
  • the separated peripheral blood lymphocyte layer may be solid-phased using at least one of anti-CD3 antibody and anti-CD161 antibody. This is because cell culture is usually performed using a flask that is solid-phased with an anti-CD3 antibody, an anti-CD161 antibody, or the like. By immobilizing with either the anti-CD3 antibody or the anti-CD161 antibody, cells contained in the peripheral blood lymphocyte layer are proliferated and activated.
  • the treatment for solid phase is not particularly limited and can be appropriately selected.
  • the method for screening cancer patients and healthy subjects of the present invention is based on the number of stained cells in the observed image in the detection step (d) in the method for detecting peripheral circulation cancer cells of the present invention described above. To do.
  • the grade is determined according to the number of stained cells in the observation image.
  • the peripheral circulating cancer cell detection device of the present invention comprises a separating means for separating lymphocytes from peripheral blood, a culturing means for culturing the separated peripheral blood lymphocyte layer in a culture solution, and a cell attached to the bottom of a culture vessel And immunostaining means for immunostaining, and detection means for detecting circulating cancer cells in the biological sample based on the observed images of the stained cells.
  • the immunostaining means is preferably stained with an anti-EMA (Epithelial Membrane Antigen) antibody.
  • the culture container used for the culture means and the immunostaining container used for the immunostaining means are the same container.
  • the culture solution may contain at least 1000 U / ml interleukin-2 (IL-2).
  • the separated peripheral blood lymphocyte layer may be immobilized using at least one of anti-CD3 antibody and anti-CD161 antibody.
  • peripheral circulating cancer cell detection method and detection apparatus of the present invention there is an effect that peripheral circulating cancer cells can be detected simply and accurately.
  • Detection flow diagram of peripheral circulation cancer cells of the present invention Detection flow chart of peripheral circulation cancer cells of the present invention (including solid phase immobilization process) EMA positive cell electron micrograph of ovarian cancer patient 1 EMA positive cell electron micrograph of ovarian cancer patient 2 EMA positive cell electron micrograph of healthy subjects 1 EMA positive cell electron micrograph of healthy subjects 2
  • the method for detecting peripheral circulation cancer cells of the present invention includes a separation step (step S1) for separating lymphocytes from peripheral blood, and culturing the separated peripheral blood lymphocyte layer in a culture solution.
  • the lymphocyte layer is separated from 30 ml of peripheral blood of the biological sample with a lymphocyte separation liquid (specific gravity 1.077 ⁇ 0.001).
  • a lymphocyte separation liquid specific gravity 1.077 ⁇ 0.001.
  • a lymphocyte culture solution prepared by adding 1000 U / ml of IL-2 to a culture solution based on RPMI1640 and IMDM in a 75 cm2 plastic flask was used. Incubate for 48 to 72 hours in an incubator at 37 ° C. with% CO 2 .
  • the proliferation rate and activity of the cells are improved.
  • IL-2 is a cytokine (immune substance released from cells) that is known to affect cell proliferation and activation, and can also be synthesized artificially.
  • the immunostaining step step S5
  • the supernatant is discarded, the cells attached to the bottom of the flask are detached with a cell scraper, fixed with 95% ethanol, and immunostained at room temperature.
  • the detection step step S7, the specimen obtained in the immunostaining step is subjected to endogenous peroxidase blocking for 5 minutes as a pretreatment, and then 6 types of antibodies described later for 20 minutes and DAB coloring reagent for 4 minutes. Hematoxylin staining is performed for 1 minute and observed under a microscope.
  • the separated lymphocyte layer is immobilized using at least one of anti-CD3 antibody and anti-CD161 antibody.
  • a process (step S2) may be added.
  • an anti-CD3 antibody or an anti-CD161 antibody, or an anti-CD3 antibody and an anti-CD161 antibody are used.
  • the reagents used in the reaction were manufactured by Dako.
  • As for hematoxylin Meyer's hematoxylin was self-adjusted and used.
  • the antibodies used in the immunostaining step (step S5) are diluted anti-EMA antibody, anti-CEA antibody, anti-CK (AE1 / AE3) antibody, anti-Ki-67 antibody, anti-CD20 (L-26) manufactured by Dako. Either an antibody or an anti-CD45RO antibody was used, and staining was performed using an automatic immunostaining apparatus. Table 1 below shows the lineage and clinical significance of cancer cells for the six types of antibodies used for immunostaining.
  • the solid-phase culture vessel was prepared by immobilizing a solid-phase treatment with anti-CD3 antibody and anti-CD161 antibody on a flask having an inner surface area of 75 cm 2 at the bottom.
  • the solid-phase treatment was performed by injecting an anti-CD3 antibody adjusted to 1 ⁇ g / ml into the flask, allowing to stand for 24 hours at room temperature, and then washing off excess antibody with a phosphate buffer.
  • the anti-CD161 antibody adjusted to 10 ⁇ g / ml was poured into the flask, allowed to stand at room temperature for 24 hours, then stored in a refrigerator at 5 ° C., and excess antibody was removed from the phosphate buffer during use. Rinse and use.
  • the step of immobilizing the separated lymphocyte layer with anti-CD3 antibody and anti-CD161 antibody may not be included.
  • Example 1 describes the result of immunostaining using an anti-EMA antibody in the immunostaining step (step S5).
  • Subject selection method As a method for selecting subjects, first, the target person is not determined, but first, the correct answer is examined, and 37 cancer patients and 22 healthy persons (including 22 males and 15 females) and 33 (of which, A total of 70 men (37 men and 33 women) were selected from 15 men and 18 women).
  • Table 2 shows the age, sex, type of cancer, stage (stage), immunostaining result (Positive: positive or Negative: negative), immunity for 37 cancer patient specimens (specimen names P1 to P37), respectively.
  • the judgment grade (0 to 3) based on the analysis result of staining is shown.
  • there are various types of cancer such as liver cancer, breast cancer, gastric cancer, pancreatic cancer, esophageal cancer, lung cancer, rectal cancer, hepatocellular carcinoma, colon cancer, and malignant lymphoma.
  • Table 3 shows the age, sex, immunostaining result (Positive: positive or Negative: negative), and determination grade (0 based on the analysis result of immunostaining for 33 healthy subjects (sample names N1 to N33), respectively. To 3).
  • sensitivity Proportion that test subjects were correctly positive
  • Specificity Proportion that non-disease was correctly negative
  • Positive predictive value Proportion of positive illness correctly
  • Negative predictive value Negative was correctly non-sick Rate that was correct
  • rate of correct diagnosis rate that was correct in the total number
  • the sensitivity, specificity, positive predictive value, negative predictive value, and correct diagnosis rate are indicators that are usually used as diagnostic accuracy. Positive rates and prevalence are not indicators of diagnostic accuracy, but are important indicators in interpreting the data. Table 5 shows calculation formulas for each index. If the sensitivity is high, there are few oversights of positive persons (prevalent), and the test has a high diagnosis probability. On the other hand, if the specificity is high, the test has few oversights (false positives). In other words, if a test with a high positive predictive value is positive, the possibility of the disease is very high, and if a test with a high negative predictive value is negative, the disease can be almost denied.
  • Anti-EMA antibodies may stain plasma cells in the bone marrow in addition to epithelial tumor cells. Since bone marrow plasma cells do not appear in peripheral blood in healthy individuals, the possibility that the healthy person's EMA-positive cells expressed in the test were plasma cells can be excluded. Cancer patients with EMA-positive cells in peripheral blood include Hodgkin's disease (H & L cells) and anaplastic large cell lymphoma (T-cell lineage cells), but these patients have not been examined this time . For these reasons, it was judged that cells stained with anti-EMA antibody can be considered epithelial tumor cells or epithelial abnormal cells.
  • H & L cells Hodgkin's disease
  • T-cell lineage cells anaplastic large cell lymphoma
  • step S3 the results of investigation using the cells of 4 healthy individuals as to whether or not the composition of the culture solution affects the expression of EMA positive cells will be described.
  • One subject was negative for EMA positive cells, and the other three were subjects who expressed EMA positive cells, and the expression of EMA positive cells was compared using culture media based on RPMI1640 and IMDM. Culturing was performed using a flask solid-phased with anti-CD3 antibody and anti-CD161 antibody. The comparison results are shown in Table 6 below. From Table 6, it can be seen that the composition of the culture solution does not affect the expression of EMA positive cells.
  • a lymphocyte layer separated from 30 ml of peripheral blood with a lymphocyte separation solution was immobilized in a flask solid-phased with an anti-CD3 antibody and an anti-CD161 antibody. It is in culture. This is because cell culture is usually performed using a flask that is solid-phased with an anti-CD3 antibody and an anti-CD161 antibody. It was investigated whether the presence or absence of the solid phase step (step S2) affects the expression of EMA positive cells. In the experiment, EMA immunostaining was performed after simultaneously culturing cells using a flask treated with anti-CD3 antibody and anti-CD161 antibody and a flask not treated with antibody.
  • EMA positive cells were expressed even when cultured in a flask that was not immobilized with the antibody, and was not related to the immobilized treatment of the antibody. That is, even if cells are cultured using an untreated flask that has not been immobilized with an antibody, the expression of EMA positive cells is not affected.
  • the presence or absence of the immobilization step (step S2) It was found that it does not affect the expression of.
  • the results of the method for detecting peripheral circulation cancer cells of this example were compared with the results measured by other methods.
  • the comparison was performed by measuring a specimen of a subject with EMA positive cells using a CellSearch (registered trademark) system (detection system for breast cancer, colon cancer, etc. developed by Veridex, USA).
  • a CellSearch registered trademark
  • Table 9 the results obtained by the EMA staining and the test results obtained by the CellSearch (registered trademark) system coincided. This indicates that the peripheral circulating cancer cell detection method of this example is not inferior to the measurement sensitivity of the CellSearch (registered trademark) system.
  • 3 and 4 are electron micrographs of EMA positive cells of ovarian cancer patients.
  • the cells observed in FIGS. 3 and 4 generally have a high N / C ratio, and formation of mitochondria and the like is observed, but formation of other cytoplasmic organelles such as the Golgi area is poor.
  • the formation of nucleolus is conspicuous. While it is difficult to recognize the formation of an intercellular adhesion device, adhesion by cytoplasmic processes is suggested. Although it is difficult to specify the cell lineage, it can be assumed that it is a tumor cell.
  • FIG. 5 and FIG. 6 are electron micrographs of EMA positive cells of a healthy person. 5 and 6, atypical cells with a high N / C ratio and conspicuous nucleoli are observed. Although some images of cell-cell adhesion are observed, the development of cell-cell adhesion devices is poor. It can be said that the growth of organelles such as mitochondria is relatively poor. From the above observation results, EMA positive cells expressed in cancer patients and healthy individuals can be inferred as some tumor cells or atypical cells, although it is difficult to specify the cell lineage. Table 10 below summarizes the comparison of observation results by electron micrographs of EMA positive cells of ovarian cancer patients and healthy individuals.
  • EMA positive cells are non-tumor stromal cells.
  • Stromal cells include immune cells, inflammatory cells, endothelial cells, fibroblasts, and pericytes. Of these, it is necessary to unravel whether EMA positive cells were stromal cells. In particular, whether it is an immune cell, inflammatory cell, or endothelial cell in the blood.
  • inflammatory cells include neutrophils and plasma cells. Neutrophils may be nonspecifically CEA positive by immunostaining, but they are not EMA positive. Plasma cells are expressed in the peripheral blood of patients with Hodgkin's disease and anaplastic large cell lymphoma, but not in the peripheral blood of healthy individuals.
  • Non-tumor immature T cells generated from thymic epithelial cells may appear in peripheral blood.
  • non-tumor immature T cells are not found in healthy individuals and appear in the peripheral blood of thymoma patients.
  • Thymoma patients are also excluded from the subject this time, so the possibility of being immune cells can be excluded.
  • endothelial cells, fibroblasts, or pericytes may be expressed as peripheral blood EMA positive cells.
  • the problems are vascular endothelial cells, fibroblasts, and pericytes that may be mixed into the needle hole during blood collection.
  • peripheral blood from which the first 2 ml was discarded at the time of blood collection and peripheral blood that was not discarded were cultured and compared.
  • EMA positive cells were expressed in peripheral blood discarded at 2 ml at the time of blood collection as well as peripheral blood not discarded at 2 ml. From this, it is possible to exclude the possibility that the detected EMA positive cells are vascular endothelial cells, fibroblasts, and pericytes mixed in the needle holes at the time of blood collection. From the above, EMA positive cells present in peripheral blood are considered epithelial cells.
  • Grade is determined as shown in Table 12 below. That is, Grade 0 is a case where there are no EMA positive cells. In this case, although there is little concern about cancer or cancer metastasis, it is recommended to undergo regular cancer screening. Grade 1 is a case where one EMA-positive cell appears. Although the probability of cancer is low, cancer screening is recommended. In this case, attention to cancer in the future is necessary.
  • Grade 2 is a case where there are several EMA positive cells, and there is a possibility of the presence of a small invisible cancer, and it is recommended to undergo a close examination of cancer.
  • Grade3 is a case where the number of EMA-positive cells is more than 5%, and in this case, it is highly recommended that cancer is present somewhere, so it is recommended to undergo a detailed examination of cancer as soon as possible. It is. Note that if the number of EMA positive cells is more than 5%, the numerical element may not be considered regardless of whether the number of EMA positive cells is 10% or 30%.
  • the healthy subject's EMA positive cells are not necessarily cancer cells relative to the EMA positive cells of cancer patients.
  • the healthy person's EMA positive cells are not necessarily cancer cells relative to the EMA positive cells of cancer patients.
  • the cancer metastasis or recurrence is significant (76%, true positive 28/37 patients), and EMA positive cells in cancer patients are cancer cells themselves. It can be said that there is a high possibility of capturing.
  • EMA-positive cells expressed in cancer patients are metastatic cancer cells, and EMA-positive cells expressed in healthy individuals may be slow-dividing cancer stem cells that may cause cancer in the future. Conceivable.
  • the possibility can be inferred from the results of observation of cultured cells of ovarian cancer patients and EMA cell-positive healthy individuals with an electron microscope (see FIGS. 3 to 6).
  • cancer patients who do not have metastasis or recurrence at the time of examination may be considered to have a higher probability of cancer metastasis or recurrence by confirming EMA positive cells after the examination.
  • cancer cells or abnormal cells are produced per day. However, these cells are eliminated by immune cells or spontaneously extinguished (apoptosis), and the onset of cancer is suppressed. If one or two of these cells remain in the body, unlike the culture condition, 100% cannot be denied unless cancer develops in vivo.
  • the result of immunostaining using an anti-CEA antibody instead of the anti-EMA antibody in the immunostaining step (step S5) will be described.
  • the anti-CEA antibody specifically stains adenocarcinoma cells such as the large intestine, lung, breast, liver, pancreas, etc., from the types of cancer and the lineage of cancer cells shown in Table 2 above, lung cancer, pancreatic cancer, large intestine Adenocarcinoma patients such as cancer and gastric cancer were selected, and the number of cases from 70 cases of Example 1 (37 cancer patients and 33 healthy persons, respectively) to 52 cases (30 cancer patients and 30 healthy persons, respectively) 22 people).
  • the results obtained by immunostaining using an anti-CEA antibody are shown in Tables 13 and 14 below. The terms and indices in the table are the same as those described in the first embodiment.
  • step S5 the result of EMA immunostaining with anti-EMA antibody and the result of EMA immunostaining with anti-CEA antibody are used at the same time.
  • the result of investigating whether or not the accuracy can be improved will be described.
  • the number of cases was reduced from 70 cases in Example 1 (37 cancer patients and 33 healthy persons, respectively) to 57 cases (35 cancer patients and 22 healthy persons, respectively).
  • the determination results obtained by simultaneously using the results of EMA immunostaining with anti-EMA antibody and the results of EMA immunostaining with anti-CEA antibody are shown in Tables 15 and 16 below. The terms and indices in the table are the same as those described in the first embodiment.
  • step S5 the result of immunostaining using an anti-Ki-67 antibody instead of the anti-EMA antibody in the immunostaining step (step S5) will be described.
  • Anti-Ki-67 antibody specifically stains cell proliferating active cells. The number of cases is 15 (8 cancer patients and 7 healthy persons, respectively). Cancer patients were randomly selected regardless of the type of cancer.
  • Tables 19 and 20 The results obtained by immunostaining using anti-Ki-67 antibody are shown in Tables 19 and 20 below. The terms and indices in the table are the same as those described in the first embodiment.
  • step S5 the result of immunostaining using an anti-CD45RO antibody instead of the anti-EMA antibody in the immunostaining step (step S5) will be described.
  • Anti-CD45RO antibody specifically stains T cell lymphoma and mature T cells. The number of cases is 13 (10 cancer patients and 3 healthy persons, respectively). Cancer patients were randomly selected regardless of the type of cancer.
  • Tables 23 and 24 The results obtained by immunostaining using an anti-CD45RO antibody are shown in Tables 23 and 24 below. The terms and indices in the table are the same as those described in the first embodiment.
  • Table 26 shows the detection accuracy when the determination by EMA immunostaining, the determination by CEA immunostaining, and the determination by using EMA immunostaining and CEA immunostaining at the same time (EMA + CEA) were obtained.
  • EMA immunostaining is most suitable as a screening test.
  • the present invention is a detection device for screening tests for early detection of cancer for healthy individuals, a device for recurrence inspection after surgery for cancer patients, and determination of the effects of anticancer agents, radiotherapy or cancer immunotherapy. Useful as a device.

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Abstract

La présente invention concerne un procédé grâce auquel une cellule tumorale circulante peut être aisément détectée avec une précision élevée. Le procédé de détection d'une cellule tumorale circulante, grâce auquel une cellule tumorale circulante dans un échantillon biologique est détectée, comprend : (a) une étape de séparation pour séparer des lymphocytes de sang périphérique ; (b) une étape de culture pour cultiver la couche de lymphocytes de sang périphérique séparés ainsi dans un milieu de culture liquide ; (c) une étape d'immunocoloration, après la culture, pour colorer immunologiquement les cellules adhérant au fond du récipient de culture ; et (d) une étape de détection pour détecter une cellule tumorale circulante dans l'échantillon biologique sur la base d'une image observée des cellules colorées qui sont obtenues dans l'étape d'immunocoloration.
PCT/JP2014/003855 2014-07-22 2014-07-22 Procédé de détection et dispositif de détection de cellule tumorale circulante Ceased WO2016013041A1 (fr)

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JP2016535556A JP6052756B2 (ja) 2014-07-22 2014-07-22 末梢循環癌細胞の検出方法および検出装置
KR1020167032240A KR101922322B1 (ko) 2014-07-22 2014-07-22 말초 순환 암세포의 검출 방법 및 검출 장치
CN201480078553.7A CN106796237B (zh) 2014-07-22 2014-07-22 末梢循环癌细胞的检测装置
PCT/JP2014/003855 WO2016013041A1 (fr) 2014-07-22 2014-07-22 Procédé de détection et dispositif de détection de cellule tumorale circulante

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JPWO2017188346A1 (ja) * 2016-04-27 2019-02-28 国立大学法人 東京大学 マイクロファイバーを用いた血中循環細胞の捕捉及び回収用材料及び当該材料を用いる方法
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CN109791152B (zh) * 2016-06-14 2023-03-03 塞勒科塔生物科学公司 用于鉴定和分离循环肿瘤细胞的磷脂醚类似物
CN106957799A (zh) * 2016-12-15 2017-07-18 郭昊伦 一种用于基于ctc循环肿瘤细胞的肿瘤精准治疗方法的装置
CN106957799B (zh) * 2016-12-15 2025-03-18 郭昊伦 一种用于基于ctc循环肿瘤细胞的肿瘤精准治疗方法的装置
JP2019060856A (ja) * 2017-09-22 2019-04-18 東ソー株式会社 目的細胞の検出方法
JP7119771B2 (ja) 2017-09-22 2022-08-17 東ソー株式会社 目的細胞の検出方法

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